The present invention relates to the field of fluorescence resonance energy transfer. In particular, the invention relates to fluorogenic assays which include a novel class of non-fluorescent quenching dyes, and to novel quenching dye compounds thereof.
Fluorescence resonance energy transfer (FRET) occurs between the electronic excited states of two fluorophores when they are in sufficient proximity to each other, in which the excited-state energy of the donor fluorophore is transferred to the acceptor fluorophore. The result is a decrease in the lifetime and a quenching of fluorescence of the donor species and a concomitant increase in the fluorescence intensity of the acceptor species. In one application of this principle, a fluorescent moiety is caused to be in close proximity to a quencher molecule. In this configuration, the energy from the excited donor fluorophore is transferred to the quencher and dissipated as heat rather than fluorescence energy.
The use of fluorescence resonance energy transfer (FRET) labels in biological systems is well known. The principle has been used in the detection of binding events or cleavage reactions in assays which employ fluorescence resonance energy transfer. In the case of peptide cleavage reactions, a fluorescent donor molecule and fluorescent acceptor molecule are attached to a peptide substrate on either side of the peptide bond to be cleaved and at such a distance that non-radiative energy transfer between the donor and the acceptor species takes place. For example, EPA 428000 discloses a novel fluorogenic peptide substrate involving a fluorescent donor molecule and a quenching acceptor molecule attached thereto. The labelled substrate can be used in the detection and assay of a viral protease enzyme, whereby, if there is enzyme present in a test sample, the substrate is cleaved and the iikonor and acceptor species are thereby separated. The resultant fluorescent emission of the donor species can be measured. Suitable fluorescent donors include fluorescein derivatives, coumarins and 5-((2-aminoethyl)amino)-naphthalene-1-sulphonic acid (EDANS). Suitable quenching acceptors include 2,4-dinitrophenyl (DNP) and 4-(4-dimethylaminophenyl)azobenzoic acid (DABCYL).
Fluorescence energy transfer has also been used in the study of nucleic acid hybridisation. For example, Tyagi and Kramer (Nature Biotechnology, 14, 303-8, (1996)) disclose homogeneous hybridisation assays which utilise fluorescent labelled probes. The hair-pin probes comprise a single-stranded nucleic acid sequence that is complementary to the target nucleic acid, together with a stem sequence formed from two complementary arms which flank the probe sequence. A fluorophore (EDANS) is attached to one arm and the non-fluorescent quencher moiety (DABCYL) is attached to the complementary arm. In the absence of target, the stem keeps the fluorescent and quenching groups in close proximity causing the fluorescence of the fluorophore to be quenched. When the probe is allowed to bind to a nucleic acid target, it undergoes a conformational change, forming a more stable hybrid with the target and forcing the arm sequences (and the fluorophore and quencher) to move apart. The fluorophore will then emit fluorescence when excited by light of a suitable wavelength.
The success of the fluorescence resonance energy transfer approach is dependent upon the choice of the appropriate donor/acceptor pair. If energy transfer between the donor and acceptor can be optimised, residual fluorescence is minimised when the donor/quencher pair are in close proximity and a large change in signal can be obtained when they are separated. There is an increasing trend towards assay miniaturisation and in high throughput screening assays and, as a result, it is beneficial to use fluorophores with high extinction coefficients in order to achieve the sensitivity levels required. A further problem associated with such assays is due to colour quenching caused by the presence in the assay medium of coloured samples which tend to absorb strongly in the 350-450 nm region of the spectrum.
The present invention provides a non-fluorescent cyanine acceptor dye that can be used as one component of a fluorescent donor/acceptor pair for assays involving the detection of binding and/or cleavage events in reactions involving biological molecules. The fluorescent donor dye possesses a high extinction coefficient, thereby enabling the detection of low levels of the fluorophore. Moreover, the fluorescent dye pair have excitation and emission wavelengths in a range which is substantially free from auto-fluorescence associated with biological samples and from quenching due to coloured samples. Additionally, the dyes are relatively pH insensitive and they possess a high degree of spectral overlap, allowing efficient energy transfer.
Accordingly, the present invention relates to a compound of formula (1): 
wherein the linker group Q contains at least one double bond and forms a conjugated system with the rings containing X and Y;
groups R3, R4, R5 and R6 are attached to the rings containing X and Y, or optionally, are attached to atoms of the Z1 and Z2 ring structures;
Z1 and Z2 each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms;
X and Y are the same or different and are selected from bis-C1-C4 alkyl- and C4-C5 spiro alkyl-substituted carbon, oxygen, sulphur, selenium, xe2x80x94CHxe2x95x90CHxe2x80x94 and Nxe2x80x94W wherein N is nitrogen and W is selected from hydrogen, a group xe2x80x94(CH2)mR8 where m is an integer from 1 to 26 and R8 is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary ammonium, nitro, primary amide, substituted amide., and groups reactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydryl groups;
at least one of groups R1, R2, R3, R4, R5, R6 and R7 is a target bonding group;
any remaining groups R3, R4, R5, R6 and R7 groups are independently selected from the group consisting of hydrogen, C1-C4 alkyl, OR9, COOR9, nitro, amino, acylamino, quaternary ammonium, phosphate, suiphonate and sulphate, where R9 is selected from H and C1-C4 alkyl;
any remaining R1 and R2 are selected from C1-C10 alkyl which may be unsubstituted or substituted with phenyl the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups;
characterised in that at least one of the groups R1, R2, R3, R4, R6, R6 and R7 comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent;
provided that the linker group Q is not a squaraine ring system.
Preferably the linker group Q contains 1, 2 or 3 double bonds in conjugation with the rings containing X and Y.
Preferably Q is the group: 
wherein the groups R10 are selected from hydrogen and C1-C4 alkyl which may be unsubstituted or substituted with phenyl, or two or more of R10 together with the group: 
form a hydrocarbon ring system substituted with R7 and which may optionally contain a heteroatom selected from xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, or  greater than NR7, wherein R7 is hereinbefore defined; and
n=1, 2 or 3.
Suitably, the non-fluorescent cyanine dye for use in the present invention is a compound having the formula (2): 
wherein groups R3, R4, R5 and R6 are attached to the rings containing X and Y or, optionally, are attached to atoms of the Z1 and Z2 ring structures and n is an integer from 1-3;
Z1 and Z2 each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms;
X and Y are the same or different and are selected from bis-C1-C4 alkyl-and C4-C5 spiro alkyl-substituted carbon, oxygen, sulphur, selenium xe2x80x94CH=CHxe2x80x94 and Nxe2x80x94W wherein N is nitrogen and W is selected from hydrogen, a group xe2x80x94(CH2)mR8 where m is an integer from 1 to 26 and R8 is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary ammonium, nitro, primary amide, substituted amide, and groups reactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydryl groups;
at least one of groups R1, R2, R3, R4, R5, R6 and R7 is a target bonding group;
any remaining groups R3, R4, R5, R6 and R7 groups are independently selected from the group consisting of hydrogen, C1-C4 alkyl, OR9, COOR9,
nitro, amino, acylamino, quaternary ammonium, phosphate, sulphonate and sulphate, where R9 is selected from H and C1-C4 alkyl;
any remaining R1 and R2 are selected from C1-C10 alkyl which may be unsubstituted or substituted with phenyl the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups;
characterised in that at least one of the groups R1, R2, R3, R4, R5, R6 and R7 comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent.
Suitably at least one of the groups R1, R2, R3, R4, R5, R6 and R7of the dyes according to the present invention comprises a substituent which reduces the fluorescence emission of the dye such that it is essentially non-fluorescent. Suitably, at least one of groups R3, R4, R5, R6 and R7 of the non-fluorescent cyanine dyes of structures (1) and (2) is a nitro group which may be attached directly to the rings containing X and Y. In the alternative, a mono- or di-nitro-substituted benzyl group may be attached to the rings containing X and Y, which optionally may be further substituted with one or more nitro groups attached directly to the aromatic rings. Preferably, at least one of groups R1, R2, R3, R4, R5, R6 and R7 of the non-fluorescent cyanine dyes of structures (1) and (2) comprises at least one nitro group.
The target bonding group R1, R2, R3, R4, R5, R6 and R7 can be any group suitable for attaching the non-fluorescent cyanine dye to a target material, such as a carrier material or a biological compound and as such will be well known to those skilled in the art. For example, the target bonding group may be a reactive group for reacting with a functional group on the target material. Alternatively, the target bonding group may be a functional group and the target may contain the reactive constituent.
Preferably, the target bonding group is of the structurexe2x80x94Exe2x80x94F where E is a spacer group and F is the reactive or functional group. A reactive group of the dye can react under suitable conditions with a functional group of a target molecule; a functional group of the dye can react under suitable conditions with a reactive group of the target molecule, whereby the target molecule becomes labelled with the dye.
Preferably, the reactive group F is selected from carboxyl, succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide, acid halide, hydrazide, vinylsulphone, dichlorotriazine and phosphoramidite. Preferably, the functional group F is selected from hydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde and ketone, phosphate and thiophosphate. By virtue of these reactive and functional groups the non-fluorescent cyanine dye may be reacted with and covalently bound to target materials.
Suitable spacer groups may contain 1-60 chain atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus. For example the spacer group may be:
xe2x80x94(CHRxe2x80x2)pxe2x80x94
xe2x80x94{(CHRxe2x80x2)qxe2x80x94Oxe2x80x94(CHRxe2x80x2)rxe2x80x94}sxe2x80x94
xe2x80x94{(CHRxe2x80x2)qxe2x80x94NRxe2x80x2xe2x80x94(CHRxe2x80x2)r}sxe2x80x94
xe2x80x94{(CHRxe2x80x2)qxe2x80x94(CHxe2x95x90CH)xe2x80x94(CHRxe2x80x2)r}sxe2x80x94
xe2x80x94{(CHRxe2x80x2)qxe2x80x94Arxe2x80x94(CHRxe2x80x2)rxe2x80x94}sxe2x80x94
xe2x80x94{(CHRxe2x80x2)qxe2x80x94COxe2x80x94NRxe2x80x2xe2x80x94(CHRxe2x80x2)rxe2x80x94}sxe2x80x94
xe2x80x94{(CHRxe2x80x2)qxe2x80x94COxe2x80x94Arxe2x80x94NRxe2x80x2xe2x80x94(CHRxe2x80x2)rxe2x80x94}sxe2x80x94
where Rxe2x80x2 is hydrogen, or C1-C4 alkyl which may be optionally substituted with suiphonate, Ar is phenylene, optionally substituted with sulphonate, xcfx81 is 1-20, preferably 1-10, q is 1-5, r is 0-5 and s is 1-5.
Specific examples of reactive groups R1-R7 and the groups with which R1-R7 can react are provided in Table 1. In the alternative, the R1-R7 may be the functional groups of Table 1 which would react with the reactive groups of a target molecule.
Particularly suitable reactive groups R1-R7 which are especially useful for labelling target components with available amino and hydroxyl functional groups include: 
where m is an integer from 1-10.
Aryl is an aromatic substituent containing one or two fused aromatic rings containing 6 to 10 carbon atoms, for example phenyl or naphthyl, the aryl being optionally and independently substituted by one or more substituents, for example halogen, straight or branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl and alkoxy for example methoxy, ethoxy, propoxy and n-butoxy.
Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ring system containing at least one and no more than 3 heteroatoms which may be selected from N, O, and S and is optionally and independently substituted by one or more substituents, for example halogen, straight or branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyl and alkoxy for example methoxy, ethoxy, propoxy and n-butoxy.
Aralkyl is a C1-C6 alkyl group substituted by an aryf or heteroaryl group.
Halogen and halo groups are selected from fluorine, chlorine, bromine and iodine.
The non-fluorescent cyanine dyes for use in the present invention may also include water solubilising constituents attached thereto for conferring a hydrophilic characteristic to the dye. They may be attached directly to the aromatic ring system of the cyanine dye or they may be attached to the spacer group E. Suitable solubilising constituents may be selected from the group consisting of sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and hydroxyl. Sulphonate or sulphonic acid groups attached directly to the aromatic ring of the non-fluorescent quenching dye are to be particularly preferred. Water solubility may be necessary when labelling proteins.
In a second aspect, the present invention relates to a biological material labelled with a non-fluorescent cyanine dye.
In a further aspect, the invention relates to a biological material which comprises two components one of which is labelled with a fluorescent dye which may act as a donor of resonance energy and the other with a non-fluorescent cyanine dye which may act as an acceptor of resonance energy transferred from the donor.
In a still further aspect, the invention relates to an assay method which comprises:
i) separating two components which are in an energy transfer relationship, the first component being labelled with a fluorescent donor dye and the second component being labelled with a non-fluorescent cyanine acceptor dye; and,
ii) detecting the presence of the first component by measuring emitted fluorescence.
In a still further aspect the invention relates to an assay method which comprises:
i) binding one component of a specific binding pair with a second component of said pair, said first component being labelled with a fluorescent donor dye and said second component being labelled with a non-fluorescent cyanine acceptor dye to bring about an energy transfer relationship between said first and second components; and,
ii) detecting the binding of the first and second components by measuring emitted fluorescence.
The non-fluorescent cyanine dyes of the present invention are employed as acceptor dyes in assay methods utilising fluorescence resonance energy transfer. When a non-fluorescent cyanine dye of the invention is in an energy transfer relationship with a fluorescent donor dye, the fluorescence emission of the donor is reduced through quenching by the acceptor. When resonance energy transfer is lost through separation of the fluorescent donor dye and the acceptor dye, the fluorescence emission due to the donor dye is restored Effective non-fluorescent quenching dyes have a low efficiency for converting absorbed incident light into fluorescence and, as such, are unsuitable as fluorescent labels where a high degree of sensitivity is required. The relative effectiveness of the non-fluorescent cyanine dyes as quencher dyes is illustrated in FIG. 1 which shows the relative fluorescence emission of representative non-fluorescent cyanine dyes of the invention compared with corresponding dyes of the same class and with similar absorption characteristics in the visible region of the spectrum.
The intrinsic fluorescence of the cyanine dyes of the present invention, when they are employed as energy acceptors is preferably less than 10% of the fluorescence emission of the donor dye upon excitation at the donor excitation wavelength and detection of emission at the donor emission wavelength. FIG. 3 shows the decrease in background signal obtainable by the use of Compound II and Compound XI as acceptor dyes compared with that of a standard matched fluorescent cyanine acceptor dye (Cy5), when measured at the emission maxima of the donor dye. The contribution to background fluorescence is thus minimised by the use of the non-fluorescent cyanine dyes of the invention as quencher dyes. Moreover, the present dyes are designed such that their spectral overlap with a fluorescent donor dye is maximised, thereby improving efficiency of quenching.
The biological material can be a biological molecule which may be cleaved into the two component parts; or the biological material may comprise two components as hereinbefore defined which may be bound either by covalent or non-covalent association.
The present invention therefore relates to a novel fluorogenic substrate and an assay method for the detection and measurement of the cleavage of a molecule into two component parts, the first component being labelled with a fluorescent donor dye in an energy transfer relationship with a non-fluorescent cyanine acceptor dye bound to the second component.
The assays may be performed according to the present invention in high throughput screening applications, including those in which compounds are to be screened for their inhibitory effects, potentiation effects, agonistic, or antagonistic effects on the reaction under investigation. Examples of such assays include, but are not restricted to, the cleavage of a peptide or protein by a protease and the cleavage of a DNA or RNA molecule by a nuclease. In this assay format, the enzyme substrate (peptide or nucleic acid) will include a sequence whose structure combines a fluorescent donor dye molecule with the non-fluorescent cyanine acceptor dye, attached to the substrate at either side of the substrate bond to be cleaved. The substrate joins the fluorescent donor and the acceptor moieties in close proximity. The intrinsic fluorescence of the donor is reduced through quenching by the acceptor due to resonance energy transfer between the pair of dyes. Resonance energy transfer becomes insignificant when the distance between the donor and acceptor moieties is greater than about 100 Angstroms. Cleavage of the substrate results in the separation between donor and acceptor dyes and concomitant loss of resonance energy transfer. The fluorescence signal of the donor fluorescent dye increases, thereby enabling accurate measurement of the cleavage reaction.
Briefly, an assay for the detection of proteolytic enzyme activity may be configured as follows. A reaction mixture is prepared by combining a protease enzyme and a fluorogenic substrate which combines a fluorescent donor dye molecule with a non-fluorescent acceptor dye of formula (2) attached to the substrate at either side of the substrate bond to be cleaved. A known or a putative protease inhibitor compound may be optionally included in the reaction mixture. Typically the reaction is performed in buffered solution and the reaction is allowed to proceed to completion. The progress of the reaction may be monitored by observing the steady state fluorescence emission due to the fluorescent donor dye, which is recorded using a spectrofluorimeter.
Alternatively, the invention relates to an assay method for detecting and measuring binding, by covalent or non-covalent association, of one component of a ligand/reactant pair with a second component of said pair, said first component being labelled with a fluorescent donor dye and said second component being labelled with a non-fluorescent cyanine acceptor dye. Such assays are conveniently categorised as one of two types.
i) The first category comprises equilibrium binding assays, in which one component of a specific binding pair binds non-covalently to a second component of the specific binding pair. Such equilibrium binding assays may be applied to screening assays in which samples containing compounds to be screened are tested for their effect upon the binding of the first component of the specific binding pair (either antagonistic or agonistic), to the second component. Either component may be labelled with the donor dye or the acceptor dye. In the absence of binding, the labelled components are too far apart for resonance energy transfer to occur. Upon binding of one labelled component to its labelled specific binding partner, the label moieties are brought into sufficiently close proximity for energy transfer to occur between donor and acceptor species resulting in a quenching of the donor fluorescence and a decrease in donor fluorescent signal.
For example, the dyes used in the present invention can be used to label probes such as those described by Tyagi and Kramer (loc. cit.) for use in the detection and identification of unique DNA sequences or specific genes in a complete DNA molecule or mixtures of nucleic acid fragments. One end of the nucleic acid probe is labelled with a fluorescent dye and at the other end with a non-fluorescent cyanine acceptor dye. In the absence of specific target sequence, the fluorescent and quenching species will be held sufficiently close for energy transfer to occur. Consequently, irradiation of the fluorophore by excitation light will give reduced fluorescent signal. Interaction of the probe with a specific target nucleic acid sequence causes a conformational change to take place in the probe, such that the fluorescent donor and acceptor become separated by distance. Excitation of the fluorophore will result in a fluorescent signal which may be recorded using a spectrofluorimeter.
Alternatively, the equilibrium binding assay may employ a sandwich assay format in which one component of a specific binding pair, such as a first antibody, is coated onto the wells of a microtitre well plate. Following binding of an antigen to the first antibody, a second antigen-specific antibody is then added to the assay mix, so as to bind with the antigen-first antibody complex; In this format, either the first antibody or the antigen may be labelled with the donor dye and the second antibody labelled with the acceptor dye or vice versa. In the absence of binding of the first antibody-antigen-second antibody complex, the labelled components are too far apart for resonance energy transfer to occur. Upon binding of the second antibody with the first antibody-antigen complex, the label moieties are brought into sufficiently close proximity for energy transfer to occur between donor and acceptor species resulting in a quenching of the donor fluorescence and a decrease in donor fluorescent signal. Fluorescence signal is measured and the concentration of antigen may be determined by interpolation from a standard curve.
Examples of specific binding pairs include, but are not restricted to, antibodies/antigens, lectins/glycoproteins, biotin/(strept)avidin, hormone/receptor, enzyme/substrate or co-factor, DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood that in the present invention, any molecules which possess a specific binding affinity may be employed, so that the energy transfer dyes of the present invention may be used for labelling one component of a specific binding pair, which in turn may be used in the detection of binding to the other component.
ii) In the second category, the assay may comprise detection and measurement of the addition of a fluorescent donor dye labelled moiety (the reactant) in solution in the assay medium to a non-fluorescent acceptor dye-labelled moiety (the substrate) or vice versa, by covalent attachment mediated through enzyme activity. Examples of such assays include, but are not restricted to, the joining of DNA or RNA molecules to other nucleic acid molecules by ligases, the addition of a nucleotide to a DNA or RNA molecule by a polymerase and the transfer of a labelled chemical moiety from one molecule to another by a transferase such as acetyl transferase. A known or a putative enzyme inhibitor may be optionally included in the reaction mixture. It is to be understood that any two appropriate reactant and substrate moieties may be employed. Either of the donor or the acceptor dyes of the present invention may be used for labelling one moiety which in turn may be used in the detection and measurement of the reaction with the substrate.
For example, in a DNA ligation assay, DNA molecules to be joined are mixed together in aqueous buffer containing ATP in the presence of a DNA ligase. Following incubation, the DNA strands are covalently attached in the correct configuration by the formation of standard phosphodiester linkages in both strands of the duplex. Upon joining the label moieties are bought into sufficiently close proximity for energy transfer to occur between donor and acceptor species resulting in a quenching of the donor fluorescence and a signal decrease which is proportional to the amount of ligated product formed.
The invention also relates to labelling methods wherein the non- fluorescent cyanine dyes of structures (1) and (2) including at least one reactive or functional group at the R1-R7 positions covalently react with amino, hydroxyl, aidehyde, phosphoryl, carboxyl, sulphydryl or other reactive groups on target materials. Such target materials include, but are not limited to the group consisting of antigen, antibody, lipid, protein, peptide, carbohydrate, nucleotides which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, and oxy or deoxy polynucleic acids which contain or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups, microbial materials, drugs and toxins.
The selection of suitable fluorescent donor and acceptor pairs is generally dependent on several factors.
i) Firstly, the donor and acceptor chromophores should have strong electronic transitions in the near UV to near IR spectral range.
ii) Secondly, the donor and acceptor moieties should be in relatively close proximity with each other. Suitably the donor and acceptor species should be in the range 10 -100 Angstroms.
iii) Thirdly, there should be a suitable overlap between the donor emission spectrum and absorption spectrum of the acceptor. The greater the overlap between donor emission spectrum and the excitation spectrum of the acceptor, the greater the energy transfer. Energy transfer can occur between dyes which share minimal spectral overlap, but this is only observed when the dyes are in close proximity.
Suitable fluorescent donor dyes that can be combined with the non-fluorescent cyanine acceptor dyes to form energy transfer pairs for the practice of the present invention include the well known reactive analogues of the fluorescein, rhodamine and cyanine dyes. Other low molecular weight fluorescent dyes may be selected from the derivatives of the bis-pyrromethine boron difluoride dyes, such as 3,3xe2x80x2,5,5xe2x80x2-tetramethyl-2,2xe2x80x2-pyrromethine-1,1xe2x80x2-boron difluoride, sold under the trademark BODIPY by Molecular Probes Inc. Particularly preferred are the cyanine dyes.
Suitable fluorescein donor dyes include: 5- and 6-carboxyfluorescein and 6-carboxy-4xe2x80x2,5xe2x80x2-dichloro-2xe2x80x2,7xe2x80x2-dimethoxyfluorescein. Suitable rhodamine dyes include: 6-carboxyrhodamine (Rhodamine 110), 5-carboxyrhodamine-6G (R6G-5 or REG-5), 6-carboxyrhodamine-6G (R6G-6 or REG-6), N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-6-carboxyrhodamine (TAMRA or TMR), 6-carboxy-X-rhodamine (ROX). Suitable cyanine donor dyes include the CyDyes(trademark): Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. (CyDye and Cy are trademarks of Amersham Pharmacia Biotech UK Limited.) Cyanine dyes suitable for use as the donor component in the assay of the present invention are disclosed in U.S. Pat. No.5268486 (Waggoner et al), or the rigidised cyanine dyes such as those disclosed in GB Patent No.2301832 (Waggoner et al). Alternatively the fluorescent donor species used as the donor component may be a fluorescence energy transfer dye cassette. Examples of such fluorescence energy transfer dye cassettes are to be found in GB Patent No.2301833 (Waggoner et al).
Table 2 below shows examples of fluorescent donor dyes and corresponding non-fluorescent cyanine acceptor dyes which are suitable for use in the methods according to the present invention.
The non-fluorescent cyanine dyes of formulae (1) and (2) may be prepared by a process comprising:
a) reacting a first compound having the formula (A): 
xe2x80x83where X, Z1, R1, R3 and R5 are hereinbefore defined,
b) a second compound which is the same or different from the first compound and having the formula (B): 
xe2x80x83where Y, Z2 R2, R4, R6 are hereinbefore defined, and
c) a third compound suitable for forming a linkage between the first and second compounds, wherein a), c) and b) are reacted either in a two-or single step process to form the compounds of formulae (1) and (2).
In the case of a two step synthesis, an intermediate dye compound is first formed by reacting an indolenine compound of structure (A) with a compound suitable for forming the linkage wherein the reaction is performed in a suitable polar solvent such as ethanol or acetic acid. In the second stage, the intermediate dye is reacted with the second indolenine compound of structure (B) in a medium such as pyridine, acetic acid and acetic anhydride at room temperature. Such reaction conditions are also suitable for the preparation of non-fluorescent cyanine dyes of the present invention by a one step process. Reagents c) which may be used for forming the linkage between the indolenine moieties are those suitable for forming a polymethine chain. Reagents and methods suitable for forming cyanine dyes containing polymethine linkages will be well known to those skilled in the art and include triethyl orthoformate, malondialdehyde bis-(phenylimine) hydrochloride and N-{(5-phenylamino)-2,4-pentadienylidene}aniline hydrochloride. (See for example, Fry D. J, Cyanine Dyes and Related Compounds, in Rodd""s Chemistry of Carbon Compounds, Elsevier 1977, page 369-422).
The non-fluorescent cyanine dyes of the present invention may be used to covalently label a target material such as a component of the assay system as hereinbefore described. Covalent labelling using compounds of the present invention may be accomplished with a target having at least one functional or reactive group as hereinbefore defined. The target may be incubated with an amount of a compound of the present invention having at least one of R1-R7 that includes a reactive or functional group as hereinbefore defined that can covalently bind with the functional or reactive group of the target material. The target material and the compound of the present invention are incubated under conditions and for a period of time sufficient to permit the target material to covalently bond to the compound of the present invention.